Antifungal agents and uses thereof

ABSTRACT

The invention features echinocandin class compounds. The compounds can be useful for the treatment of fungal infections.

BACKGROUND OF THE INVENTION

This invention relates to the field of treatment of fungal infections.

The need for novel antifungal treatments is significant, and isespecially critical in the medical field. Immunocompromised patientsprovide perhaps the greatest challenge to modern health care delivery.During the last three decades there has been a dramatic increase in thefrequency of fungal infections in these patients (Herbrecht, Eur. J.Haematol., 56:12, 1996; Cox et al., Curr. Opin. Infect. Dis., 6:422,1993; Fox, ASM News, 59:515, 1993). Deep-seated mycoses are increasinglyobserved in patients undergoing organ transplants and in patientsreceiving aggressive cancer chemotherapy (Alexander et al., Drugs,54:657, 1997). The most common pathogens associated with invasive fungalinfections are the opportunistic yeast, Candida albicans, and thefilamentous fungus, Aspergillus fumigants (Bow, Br. J. Haematol., 101:1,1998; Wamock, J. Antimicrob. Chemother., 41:95, 1998). There are anestimated 200,000 patients per year who acquire nosocomial fungalinfections (Beck-Sague et al., J. Infect. Dis., 167:1247, 1993). Alsoadding to the increase in the numbers of fungal infections is theemergence of Acquired Immunodeficiency Syndrome (AIDS) where virtuallyall patients become affected with some form of mycoses during the courseof the disease (Alexander et al., Drugs, 54:657, 1997; Hood et al., J.Antimicrob. Chemother., 37:71, 1996). The most common organismsencountered in these patients are Cryptococcus neoformans, Pneumocystiscarinii, and C. albicans (HIV/AIDS Surveillance Report, 1996, 7(2),Year-End Edition; Polis, M. A. et al., AIDS: Biology, Diagnosis,Treatment and Prevention, fourth edition, 1997). New opportunisticfungal pathogens such as Penicillium marneffei, C. krusei, C. glabrata,Histoplasma capsulatum, and Coccidioides immitis are being reported withregularity in immunocompromised patients throughout the world.

The development of antifungal treatment regimens has been a continuingchallenge. Currently available drugs for the treatment of fungalinfections include amphotericin B, a macrolide polyene that interactswith fungal membrane sterols, flucytosine, a fluoropyrimidine thatinterferes with fungal protein and DNA biosynthesis, and a variety ofazoles (e. g., ketoconazole, itraconazole, and fluconazole) that inhibitfungal membrane-sterol biosynthesis (Alexander et al., Drugs, 54:657,1997). Even though amphotericin B has a broad range of activity and isviewed as the “gold standard” of antifungal therapy, its use is limiteddue to infusion-related reactions and nephrotoxicity (Warnock, J.Antimicrob. Chemother., 41:95, 1998). Flucytosine usage is also limiteddue to the development of resistant microbes and its narrow spectrum ofactivity. The widespread use of azoles is causing the emergence ofclinically-resistant strains of Candida spp. Due to the problemsassociated with the current treatments, there is an ongoing search fornew treatments.

When the echinocandin caspofungin was approved for sale in 2001, itrepresented the first new class of antifungal agents to be approved inover a decade. Since that time, two other echinocandin antifungals,anidulafungin and micafungin, have been approved in various markets.Each agent in this class of compound acts by inhibition of β-1,3-glucansynthase, which is a key enzyme in the synthesis of glucan in the cellwall of many fungi. All three of these drugs are made semisynthetically,starting with natural products obtained through fermentation.

The echinocandins are a broad group of antifungal agents that typicallyare comprised of a cyclic hexapeptide and lipophilic tail, the latter ofwhich is attached to the hexapeptide core through an amide linkage.Although many echinocandins are natural products, the clinicallyrelevant members of this class have all been semisynthetic derivatives.Although the naturally occurring echinocandins possess some degree ofanti-fungal activity, they have not been suitable as therapeutics,primarily because of poor aqueous solubility, insufficient potency,and/or hemolytic action. The approved echinocandins are the products ofintense efforts to generate derivatives that maintain or improve uponthe glucan synthase inhibition, but do not cause the hemolytic effects.As therapeutic agents, they are attractive compounds in terms of theirsystemic half-lives, large therapeutic windows, safety profiles, andrelative lack of interactions with other drugs. Unfortunately, the pooraqueous solubility and poor intestinal absorption of these compoundshave relegated them to delivery by intravenous infusion. Althoughpatients receiving these drugs are often hospitalized with seriousinfections, the ability to transition patients from intravenous deliveryin a hospital setting to oral delivery in a home setting would be verydesirable, especially considering the course of the regimen commonlyexceeds 14 days. In addition, an oral echinocandin may expand the use ofthis drug class to include patients that present with mild fungalinfections.

SUMMARY OF THE INVENTION

The present invention features derivatives of echinocandin antifungalsthat can have increased aqueous solubility. More specifically, theinvention features echinocandin class compounds that have been modifiedsuch that they can exhibit (i) activity against one or more fungalspecies or genera; (ii) increased aqueous solubility and/oramphiphilicity; (iii) have an increased therapeutic index; (iv)suitability for topical administration; (v) suitability for intravenousadministration; (vi) have an increased volume of distribution; and/or(vii) have an increased elimination half-life.

The invention features compounds of formula (I):

In formula (I), R¹ is O(CH₂CH₂O)CH₂CH₂X₁, O(CH₂CH₂CH₂O)CH₂CH₂X₁,NHCH₂CH₂X₂, NH(CH₂CH₂O)_(m)CH₂CH₂X₂, NH(CH₂CH₂CH₂O)_(m)CH₂CH₂X₂,NH(CH₂CH₂O)_(p)CH₂CH₂X₃, NH(CH₂CH₂CH₂O)_(p)CH₂CH₂X₃, NHCH₂CH₂X₄,NH[CH₂(CH₂)_(a)O]_(b)CH{CH₂[OCH₂(CH₂)_(c)]_(d)X₅}₂,O[CH₂(CH₂)_(a)O]_(b)CH{CH₂[OCH₂(CH₂)_(c)]_(d)X₅}₂,NH(CH₂CH₂NH)_(r)CH₂CH₂X₅, NHCH₂(CH₂)_(q)X₆, or OCH₂(CH₂)_(q)X₆; R^(T) isn-pentyl, sec-pentyl, or iso-pentyl; X₁ is NH₂, NHR^(A1), NR^(A1)R^(A2),NR^(A1)R^(A2)R^(A3), or NHCH₂(CH₂)_(v)Z₁; X₂ is OH, OR^(B1), orOCH₂(CH₂)_(v)Z₁; X₃ is NH₂, NHR^(C1), NR^(C1)R^(C2), orNR^(C1)R^(C2)R^(C3), or NHCH₂(CH₂)_(v)Z₁; X₄ is NR^(D1)R^(D2)R^(D3) orNHCH₂(CH₂)_(v)Z₁; each X₅ is, independently, selected from OH, OR^(E1),NH₂, NHR^(E1), NR^(E1)R^(E2), NR^(E1)R^(E2), R^(E3), OCH₂(CH₂)_(v)Z₁,and NHCH₂(CH₂)_(v)Z₁; X₆ is selected from NR^(F1)R^(F2)R^(F3) or Z₁; ais an integer from 1 to 2; b is an integer from 0 to 3 (e.g., 0, 1, 2,or 3); c is an integer from 1 to 2; d is an integer from 0 to 3 (e.g.,0, 1, 2, or 3); n is an integer from 1 to 5 (e.g., 1, 2, 3, 4, or 5); mis an integer from 1 to 5 (e.g., 1, 2, 3, 4, or 5); p is an integer from1 to 5 (e.g., 1, 2, 3, 4, or 5); r is an integer from 1 to 5 (e.g., 1,2, 3, 4, or 5); q is an integer from 1 to 3 (e.g., 1, 2, or 3); v is aninteger from 1 to 3 (e.g., 1, 2, or 3); each of R^(A1), R^(A2), R^(A3),R^(B1), R^(C1), R^(C2), R^(C3), R^(D1), R^(D2), R^(D3), R^(E1), R^(E2),R^(E3), R^(F1), R^(F2), and R^(F3) is, independently, selected from CH₃,CH₂CH₃, CH₂CH₂CH₃, and CH(CH₃)₂; Z₁ is selected from:

and each of R^(1A), R^(2A), R^(3A), R^(4A), R^(5A), R^(6A), R^(7A),R^(8A), R^(9A), R^(10A), R^(11A), R^(12A), R^(13A), R^(14A), R^(15A),R^(16A), R^(17A), R^(18A), R^(19A), R^(20A), R^(21A), and R^(22A), isindependently, selected from H, CH₃, CH₂CH₃, CH₂CH₂CH₃, and CH(CH₃)₂, ora pharmaceutically acceptable salt thereof.

In certain embodiments, the compound of formula (I) is further describedby formula (Ia):

In formula (Ia), R¹ is O(CH₂CH₂O)_(n)CH₂CH₂X₁, O(CH₂CH₂CH₂O)₆CH₂CH₂X₁,NHCH₂CH₂X₂, NH(CH₂CH₂O)_(p)CH₂CH₂X₂, NH(CH₂CH₂CH₂O)_(m)CH₂CH₂X₂,NH(CH₂CH₂O)_(p)CH₂CH₂X₃, NH(CH₂CH₂CH₂O)_(p)CH₂CH₂X₃, NHCH₂CH₂X₄,NH[CH₂(CH₂)_(a)O]_(b)CH{CH₂[OCH₂(CH₂)_(c)]_(d)X₅}₂,O[CH₂(CH₂)_(a)O]_(b)CH{CH₂[OCH₂(CH₂)_(c)]_(d)X₅}₂,NH(CH₂CH₂NH)_(r)CH₂CH₂X₅, NHCH₂(CH₂)_(q)X₆, or OCH₂(CH₂)_(q)X₆; R^(T) isn-pentyl, sec-pentyl, or iso-pentyl; X₁ is NH₂, NHR^(A1), NR^(A1)R^(A2),or NR^(A1)R^(A2)R^(A3); X₂ is OH or OR^(B1); X₃ is NH₂, NHR^(C1),NR^(C1)R^(C2), or NR^(C1)R^(C2)R^(C3); X₄ is NR^(D1)R^(D2)R^(D3); eachX₅ is, independently, selected from OH, OR^(E1), NH₂, NHR^(E1),NR^(E1)R^(E2), and NR^(E1)R^(E2)R^(E3); X₆ is selected fromNR^(F1)R^(F2)R^(F3); a is an integer from 1 to 2; b is an integer from 0to 3 (e.g., 0, 1, 2, or 3); c is an integer from 1 to 2; d is an integerfrom 0 to 3 (e.g., 0, 1, 2, or 3); n is an integer from 1 to 5 (e.g., 1,2, 3, 4, or 5); m is an integer from 1 to 5 (e.g., 1, 2, 3, 4, or 5); pis an integer from 1 to 5 (e.g., 1, 2, 3, 4, or 5); r is an integer from1 to 5 (e.g., 1, 2, 3, 4, or 5); q is an integer from 1 to 3 (e.g., 1,2, or 3); and each of R^(A1), R^(A2), R^(A3), R^(B1), R^(C1), R^(C2),R^(C3), R^(D1), R^(D2), R^(D3), R^(E1), R^(E2), R^(E3), R^(F1), R^(F2),and R^(F3) is, independently, selected from CH₃, CH₂CH₃, CH₂CH₂CH₃, andCH(CH₃)₂, or a pharmaceutically acceptable salt thereof. In particularembodiments of the compounds of formula (I) and (Ia), one of X₁, X₃, X₄,and X₅ is selected from N(CH₃)₃ ⁺ and N(CH₂CH₃)₃ ^(T). In certainembodiments of the compounds of formula (I) and (Ia), R¹ isNHCH[CH₂CH₂N(CH₃)₃ ⁺]₂, NHCH₂CH₂OCH[CH₂CH₂N(CH₃)₃ ⁺]₂, orNHCH₂CH₂OCH[CH₂CH₂N(CH₃)₃ ⁺][CH₂CH₂OCH₂CH₂OH].

In still other embodiments, the compound of formula (I) is furtherdescribed by formula (Ib):

In formula (Ib), R¹ is O(CH₂CH₂O)CH₂CH₂X₁, O(CH₂CH₂CH₂O)CH₂CH₂X₁,NHCH₂CH₂X₂, NH(CH₂CH₂O)_(m)CH₂CH₂X₂, NH(CH₂CH₂CH₂O)_(m)CH₂CH₂X₂,NH(CH₂CH₂O)_(p)CH₂CH₂X₃, NH(CH₂CH₂CH₂O)_(p)CH₂CH₂X₃, NHCH₂CH₂X₄,NH[CH₂(CH₂)_(a)O]_(b)CH{CH₂[OCH₂(CH₂)_(c)]_(d)X₅}₂,O[CH₂(CH₂)_(a)O]_(b)CH{CH₂[OCH₂(CH₂)_(c)]_(d)X₅}₂,NH(CH₂CH₂NH)_(r)CH₂CH₂X₅, NHCH₂(CH₂)_(q)X₆, or OCH₂(CH₂)_(q)X₆; R^(T) isn-pentyl, sec-pentyl, or iso-pentyl; X₁ is NHCH₂(CH₂)_(v)Z₁; X₂ isOCH₂(CH₂)_(v)Z₁; X₃ is NHCH₂(CH₂)_(v)Z₁; X₄ is NHCH₂(CH₂)_(v)Z₁; each X₅is, independently, selected from OCH₂(CH₂)_(v)Z₁ and NHCH₂(CH₂)_(v)Z₁;X₆ is Z₁; a is an integer from 1 to 2; b is an integer from 0 to 3(e.g., 0, 1, 2, or 3); c is an integer from 1 to 2; d is an integer from0 to 3 (e.g., 0, 1, 2, or 3); n is an integer from 1 to 5 (e.g., 1, 2,3, 4, or 5); m is an integer from 1 to 5 (e.g., 1, 2, 3, 4, or 5); p isan integer from 1 to 5 (e.g., 1, 2, 3, 4, or 5); r is an integer from 1to 5 (e.g., 1, 2, 3, 4, or 5); q is an integer from 1 to 3 (e.g., 1, 2,or 3); v is an integer from 1 to 3 (e.g., 1, 2, or 3); Z₁ is selectedfrom:

and each of R^(1A), R^(2A), R^(3A), R^(4A), R^(5A), R^(6A), R^(7A),R^(8A), R^(9A), R^(10A), R^(11A), R^(12A), R^(13A), R^(14A), R^(15A),R^(16A), R^(17A), R^(18A), R^(19A), R^(20A), R^(21A), and R^(22A), is,independently, selected from H, CH₃, CH₂CH₃, CH₂CH₂CH₃, and CH(CH₃)₂, ora pharmaceutically acceptable salt thereof.

In one particular embodiment of the compounds of formula (I), (Ia), and(Ib), the compound is further described by one of the formulas:

wherein R¹ and R^(T) are as described above.

The invention further features compounds of formula (II):

In formula (II), R² is NH(CH₂CH₂O)_(s)CH₂CH₂X₈,NH(CH₂CH₂CH₂O)_(s)CH₂CH₂X₈, NH(CH₂CH₂NH)_(t)CH₂CH₂X₉,NH[CH₂(CH₂)_(a)O]_(b)CH {CH₂[OCH₂(CH₂)_(c)]_(d)X₉}₂,O[CH₂(CH₂)_(a)O]_(b)CH{CH₂[OCH₂(CH₂)_(c)]_(d)X₉}₂, NHCH₂(CH₂)_(u)X₁₀, orOCH₂(CH₂)_(u)X₁₀; X₈ is OH, OR^(G1), NH₂, NHR^(G1), NR^(G1)R^(G2),NR^(G1)R^(G2)R^(G3), OCH₂(CH₂)_(w)Z₂, or NHCH₂(CH₂)_(v)Z₂; each X₉ is,independently, selected from OH, OR^(H1), NHR^(H1), NR^(H1)R^(H2),NR^(H1)R^(H2)R^(H3), OCH₂(CH₂)_(w)Z₂, and NHCH₂(CH₂)_(v)Z₂; X₁₀ isselected from NR^(I1)R^(I2)R^(I3) or Z₂; a is an integer from 1 to 2; bis an integer from 0 to 3 (e.g., 0, 1, 2, or 3); c is an integer from 1to 2; d is an integer from 0 to 3 (e.g., 0, 1, 2, or 3); s is an integerfrom 1 to 5 (e.g., 1, 2, 3, 4, or 5); t is an integer from 1 to 5 (e.g.,1, 2, 3, 4, or 5); u is an integer from 1 to 3 (e.g., 1, 2, or 3); eachof R^(G1), R^(G2), R^(G3), R^(H1), R^(H2), R^(H3), R^(I1), R^(I2), andR^(I3) is, independently, selected from CH₃, CH₂CH₃, CH₂CH₂CH₃, andCH(CH₃)₂; w is an integer from 1 to 3 (e.g., 1, 2, or 3); Z₂ is selectedfrom

and each of R^(1A), R^(2A), R^(3A), R^(4A), R^(5A), R^(6A), R^(7A),R^(8A), R^(9A), R^(10A), R^(11A), R^(12A), R^(13A), R^(14A), R^(15A),R^(16A), R^(17A), R^(18A), R^(19A), R^(20A), R^(21A), and R^(22A), is,independently, selected from H, CH₃, CH₂CH₃, CH₂CH₂CH₃, and CH(CH₃)₂, ora pharmaceutically acceptable salt thereof.

In certain embodiments, the compound of formula (II) is furtherdescribed by formula (IIa):

In formula (IIa), R² is NH(CH₂CH₂O)_(s)CH₂CH₂X₈,NH(CH₂CH₂CH₂O)_(s)CH₂CH₂X₈, NH(CH₂CH₂NH)_(t)CH₂CH₂X₉, NH[CH₂(CH₂)_(a)O]_(b)CH{CH₂[OCH₂(CH₂)]_(d)X₉}₂,O[CH₂(CH₂)_(a)O]_(b)CH{CH₂[OCH₂(CH₂)_(c)]_(d)X₉}₂, NHCH₂(CH₂)_(u)X₁₀, orOCH₂(CH₂)_(u)X₁₀; X₈ is OH, OR^(G1) NH₂, NHR^(G1), NR^(G1)R^(G2), orNR^(G1)R^(G2)R^(G3); each X₉ is, independently, selected from OH,OR^(H1), NHR^(H1), NR^(H1)R^(H2), and NR^(H1)R^(H2)R^(H3); X₁₀ isselected from N^(I1)R^(I2)R^(I3); a is an integer from 1 to 2; b is aninteger from 0 to 3 (e.g., 0, 1, 2, or 3); c is an integer from 1 to 2;d is an integer from 0 to 3 (e.g., 0, 1, 2, or 3); s is an integer from1 to 5 (e.g., 1, 2, 3, 4, or 5); t is an integer from 1 to 5 (e.g., 1,2, 3, 4, or 5); u is an integer from 1 to 3 (e.g., 1, 2, or 3); and eachof R^(G1), R^(G2), R^(G3), R^(H1), R^(H2), R^(H3), R^(I1), R^(I2), andR^(I3) is, independently, selected from CH₃, CH₂CH₃, CH₂CH₂CH₃, andCH(CH₃)₂, or a pharmaceutically acceptable salt thereof. In particularembodiments of the compounds of formula (II) and (IIa), one of X₈ and X₉is selected from N(CH₃)₃ ⁺ and N(CH₂CH₃)₃ ⁺. In certain embodiments ofthe compounds of formula (II) and (IIa), R² is NHCH[CH₂CH₂N(CH₃)₃ ⁺]₂,NHCH₂CH₂OCH[CH₂CH₂N(CH₃)₃ ⁺]₂, or NHCH₂CH₂OCH[CH₂CH₂N(CH₃)₃⁺][CH₂CH₂OCH₂CH₂OH].

In still other embodiments, the compound of formula (I) is furtherdescribed by formula (IIb):

In formula (IIb), R² is NH(CH₂CH₂O)_(s)CH₂CH₂X₈,NH(CH₂CH₂CH₂O)_(s)CH₂CH₂X₈, NH(CH₂CH₂NH)_(t)CH₂CH₂X₉,NH[CH₂(CH₂)_(a)O]_(b)CH {CH₂[OCH₂(CH₂)_(c)]_(d)X₉}₂,O[CH₂(CH₂)_(a)O]_(b)CH{CH₂[OCH₂(CH₂)_(c)]_(d)X₉}₂, NHCH₂(CH₂)_(u)X₁₀, orOCH₂(CH₂)_(u)X₁₀; X₈ is OCH₂(CH₂)_(w)Z₂ or NHCH₂(CH₂)_(v)Z₂; each X₅ is,independently, selected from OCH₂(CH₂)_(w)Z₂ and NHCH₂(CH₂)_(v)Z₂; X₁₀is Z₂; a is an integer from 1 to 2; b is an integer from 0 to 3 (e.g.,0, 1, 2, or 3); c is an integer from 1 to 2; d is an integer from 0 to 3(e.g., 0, 1, 2, or 3); s is an integer from 1 to 5 (e.g., 1, 2, 3, 4, or5); t is an integer from 1 to 5 (e.g., 1, 2, 3, 4, or 5); u is aninteger from 1 to 3 (e.g., 1, 2, or 3); w is an integer from 1 to 3(e.g., 1, 2, or 3); Z₂ is selected from

and each of R^(1A), R^(2A), R^(3A), R^(4A), R^(5A), R^(6A), R^(7A),R^(8A), R^(9A), R^(10A), R^(11A), R^(12A), R^(13A), R^(14A), R^(15A),R^(16A), R^(17A), R^(18A), R^(19A), R^(20A), R^(21A), and R^(22A), is,independently, selected from H, CH₃, CH₂CH₃, CH₂CH₂CH₃, and CH(CH₃)₂, ora pharmaceutically acceptable salt thereof.

In one particular embodiment of the compounds of formula (II), (IIa),and (IIb), the compound is further described by one of the formulas:

wherein R² is as described above.

The invention also features compounds described by formula (III):

In formula (III), R¹ is O(CH₂CH₂O)CH₂CH₂X₁, O(CH₂CH₂CH₂O)CH₂CH₂X₁,NHCH₂CH₂X₂, NH(CH₂CH₂O)_(m)CH₂CH₂X₂, NH(CH₂CH₂CH₂O)_(m)CH₂CH₂X₂,NH(CH₂CH₂O)_(p)CH₂CH₂X₃, NH(CH₂CH₂CH₂O)_(p)CH₂CH₂X₃, NHCH₂CH₂X₄,NH[CH₂(CH₂)_(a)O]_(b)CH{CH₂[OCH₂(CH₂)_(c)]_(d)X₅}₂,O[CH₂(CH₂)_(a)O]_(b)CH{CH₂[OCH₂(CH₂)_(c)]_(d)X₅}₂,NH(CH₂CH₂NH)_(r)CH₂CH₂X₅, NHCH₂(CH₂)_(q)X₆, or OCH₂(CH₂)_(q)X₆; R² is H,CH₃, CH₂CH₂NH₂, or CH₂C(O)NH₂; R^(T) is n-pentyl, sec-pentyl, oriso-pentyl; X is NH₂, NHR^(A1), NR^(A1)R^(A2), NR^(A1)R^(A2)R^(A3), orNHCH₂(CH₂)_(v)Z₁; X₂ is OH, OR^(B1), or OCH₂(CH₂)_(v)Z₁; X₃ is NH₂,NHR^(C1), NR^(C1)R^(C2), or NR^(C1)R^(C2)R^(C3), or NHCH₂(CH₂)_(v)Z₁; X₄is NR^(D1)R^(D2)R^(D3) or NHCH₂(CH₂)_(v)Z₁; each X₅ is, independently,selected from OH, OR^(E1), NH₂, NHR^(E1), NR^(E1)R^(E2),NR^(E1)R^(E2)R^(E3), OCH₂(CH₂)_(v)Z₁, and NHCH₂(CH₂)_(v)Z₁; X₆ isselected from NR^(F1)R^(F2)R^(F3) or Z₁; a is an integer from 1 to 2; bis an integer from 0 to 3 (e.g., 0, 1, 2, or 3); c is an integer from 1to 2; d is an integer from 0 to 3 (e.g., 0, 1, 2, or 3); n is an integerfrom 1 to 5 (e.g., 1, 2, 3, 4, or 5); m is an integer from 1 to 5 (e.g.,1, 2, 3, 4, or 5); p is an integer from 1 to 5 (e.g., 1, 2, 3, 4, or 5);r is an integer from 1 to 5 (e.g., 1, 2, 3, 4, or 5); q is an integerfrom 1 to 3 (e.g., 1, 2, or 3); v is an integer from 1 to 3 (e.g., 1, 2,or 3); each of R^(A1), R^(A2), R^(A3), R^(B1), R^(C1), R^(C2), R^(C3),R^(D1), R^(D2), R^(D3), R^(E1), R^(E2), R^(E3), R^(F1), R^(F2), andR^(F3) is, independently, selected from CH₃, CH₂CH₃, CH₂CH₂CH₃, andCH(CH₃)₂; Z₁ is selected from:

and each of R^(1A), R^(2A), R^(3A), R^(4A), R^(5A), R^(6A), R^(7A),R^(8A), R^(9A), R^(10A), R^(11A), R^(12A), R^(13A), R^(14A), R^(15A),R^(16A), R^(17A), R^(18A), R^(19A), R^(20A), R^(21A), and R^(22A), is,independently, selected from H, CH₃, CH₂CH₃, CH₂CH₂CH₃, and CH(CH₃)₂, ora pharmaceutically acceptable salt thereof. In particular embodiments ofthe compounds of formula (III), one of X₁, X₃, X₄, X₅, and X₆ isselected from N(CH₃)₃ ⁺ and N(CH₂CH₃)₃ ⁺.

In one particular embodiment of the compounds of formula (III), thecompound is further described by one of the formulas:

wherein R¹ and R^(T) are as described above.

The compounds of the invention include, without limitation, compound 1,compound 2, compound 3, compound 4, compound 5, compound 6, compound 7,compound 8, compound 9, compound 10, compound 11, compound 12, compound13, compound 14, compound 16, compound 17, compound 18, compound 19,compound 20, compound 21, compound 22, and salts thereof.

The compounds of the invention can increased amphiphilicity; increasedaqueous solubility (e.g., in 0.1M acetate buffer at pH 5.6); anincreased therapeutic index; an increased elimination half-life; and/oran increased volume of distribution.

The invention also features a pharmaceutical composition including acompound of the invention, or a pharmaceutically acceptable saltthereof, and a pharmaceutically acceptable excipient. In particularembodiments, the pharmaceutical composition includes an acetate salt ora chloride salt of a compound of the invention.

The pharmaceutical compositions of the invention can be formulated forintravenous, topical, or oral administration in unit dosage form, or anyother dosage form described herein.

The invention further features a method of treating a fungal infectionin a subject by administering to the subject a pharmaceuticalcomposition of the invention in an amount sufficient to treat theinfection. In particular embodiments, the pharmaceutical composition isadministered intravenously or topically. The pharmaceutical compositioncan be administered to treat a blood stream infection, tissue infection(e.g., lung, kidney, or liver infection) in the subject, or any othertype of infection described herein. The fungal infection being treatedcan be an infection selected from tinea capitis, tinea corporis, tineapedis, onychomycosis, perionychomycosis, pityriasis versicolor, oralthrush, vaginal candidosis, respiratory tract candidosis, biliarycandidosis, eosophageal candidosis, urinary tract candidosis, systemiccandidosis, mucocutaneous candidosis, aspergillosis, mucormycosis,paracoccidioidomycosis, North American blastomycosis, histoplasmosis,coccidioidomycosis, sporotrichosis, fungal sinusitis, or chronicsinusitis. In certain embodiments, the infection being treated is aninfection by Candida albicans, C. parapsilosis, C. glabrata, C.guilliermondii, C. krusei, C. lusitaniae, C. tropicalis, Aspergillusfumigatus, A. flavus, A. terreus. A. niger, A. candidus, A. clavatus, orA. ochraceus.

The invention features a method of preventing a fungal infection in asubject by administering to the subject a pharmaceutical composition ofthe invention in an amount sufficient to prevent the infection. Inparticular embodiments, the pharmaceutical composition is administeredintravenously at least once over a period of 1-30 days (e.g., 1, 2, 3,4, or 5 times over a period of 1-30 days). For example, the methods ofthe invention can be used for prophylaxis treatment in subjects beingprepared for an invasive medical procedure (e.g., preparing for surgery,such as receiving a transplant, stem cell therapy, a graft, aprosthesis, receiving long-term or frequent intravenous catheterization,or receiving treatment in an intensive care unit), in immunocompromisedsubjects (e.g., subjects with cancer, with HIV/AIDS, or takingimmunosuppressive agents), or in subjects undergoing long termantibiotic therapy.

In one particular embodiment of any of the methods of the invention, thepharmaceutical composition includes compound 1, or any other compounddescribed herein, or a pharmaceutically acceptable salt thereof.

The invention also features a method of preventing, stabilizing, orinhibiting the growth of fungi, or killing fungi by contacting the fungior a site susceptible to fungal growth with a compound of the invention,or a pharmaceutically acceptable salt thereof.

As used herein, the terms “an amount sufficient” and “sufficient amount”refer to the amount of a drug required to treat or prevent an infection.The sufficient amount used to practice the invention for therapeutic orprophylactic treatment of conditions caused by or contributed to by aninfection varies depending upon the manner of administration, the typeof infection, the age, body weight, and general health of the subject.Ultimately, the attending physician or veterinarian will decide theappropriate amount and dosage regimen. Such amount is referred to as a“sufficient” amount.

By “fungal infection” is meant the invasion of a host by pathogenicfungi. For example, the infection may include the excessive growth offungi that are normally present in or on the body of a subject or growthof fungi that are not normally present in or on a subject. Moregenerally, a fungal infection can be any situation in which the presenceof a fungal population(s) is damaging to a host body. Thus, a subject is“suffering” from a fungal infection when an excessive amount of a fungalpopulation is present in or on the subject's body, or when the presenceof a fungal population(s) is damaging the cells or other tissue of thesubject.

By “increased amphiphilicity” is meant an increase in the solubility ofa compound of the invention in both water (0.1M acetate buffer at pH5.6) and glycerol in comparison to the parent echinocandin compound(i.e., compounds of formula (I), (Ia), and (Ib) can have an increasedamphiphilicity in comparison to anidulafungin; compounds of formula(II), (IIa), and (IIb) can have an increased amphiphilicity incomparison to caspofungin; and compounds of formula (III) can have anincreased amphiphilicity in comparison to micafungin).

By “increased elimination half-life” is meant an increase in theelimination half-life (e. g., as observed in a PK study as described inExample 24) for a compound of the invention in comparison to the parentechinocandin compound (i.e., compounds of formula (I), (Ia), and (Ib)can have an increased elimination half-life in comparison toanidulafungin; compounds of formula (II), (IIa), and (IIb) can have anincreased elimination half-life in comparison to caspofungin; andcompounds of formula (III) can have an increased elimination half-lifein comparison to micafungin) administered under the same conditions(e.g., with the same carriers and other inactive excipients and by thesame route). The compounds of the invention can exhibit at least 25%,50%, 100%, 200%, or 300% longer elimination half-life than thecorresponding parent echinocandin class compound.

By “increased volume of distribution” is meant an increase in the volumeof distribution (e. g., as observed in a PK study as described inExample 24) for a compound of the invention in comparison to the parentechinocandin compound (i.e., compounds of formula (I), (Ia), and (Ib)can have an increased volume of distribution in comparison toanidulafungin; compounds of formula (II), (IIa), and (IIb) can have anincreased volume of distribution in comparison to caspofungin; andcompounds of formula (III) can have an increased volume of distributionin comparison to micafungin) administered under the same conditions(e.g., with the same carriers and other inactive excipients and by thesame route). The compounds of the invention can exhibit at least 25%,50%, 100%, 200%, or 300% greater volume of distribution than thecorresponding parent echinocandin class compound.

By “increased therapeutic index” is meant an increase in the ratio ofmedian lethal dose (LD₅₀) to median effective dose (ED⁵⁰) (e. g., asobserved using a mouse model of infection) for a compound of theinvention in comparison to the parent echinocandin compound (i.e.,compounds of formula (I), (Ia), and (Ib) can have an increasedtherapeutic index in comparison to anidulafungin; compounds of formula(II), (IIa), and (IIb) can have an increased therapeutic index incomparison to caspofungin; and compounds of formula (III) can have anincreased therapeutic index in comparison to micafungin) administeredunder the same conditions (e.g., with the same carriers and otherinactive excipients and by the same route). The compounds of theinvention can exhibit at least 25%, 50%, 100%, 200%, or 300% greatertherapeutic index than the corresponding parent echinocandin classcompound. For example, the compounds of the invention can exhibitextended circulating half-lives in vivo, allowing similar efficacy to beachieved at lower doses for the compound of the invention in comparisonto the parent echinocandin compound.

As used herein, the term “treating” refers to administering apharmaceutical composition for prophylactic and/or therapeutic purposes.To “prevent disease” refers to prophylactic treatment of a subject whois not yet ill, but who is susceptible to, or otherwise at risk of, aparticular disease. To “treat disease” or use for “therapeutictreatment” refers to administering treatment to a subject alreadysuffering from a disease to improve or stabilize the subject'scondition. Thus, in the claims and embodiments, treating is theadministration to a subject either for therapeutic or prophylacticpurposes.

The term “unit dosage form” refers to physically discrete units suitableas unitary dosages, such as a pill, tablet, caplet, hard capsule or softcapsule, each unit containing a predetermined quantity of a drug. By“hard capsule” is meant a capsule that includes a membrane that forms atwo-part, capsule-shaped, container capable of carrying a solid orliquid payload of drug and excipients. By “soft capsule” is meant acapsule molded into a single container carrying a liquid or semisolidpayload of drug and excipients.

Other features and advantages of the invention will be apparent from thefollowing detailed description, the drawings, and the claims.

DRAWINGS

FIG. 1 is a table of MEC and MIC values versus Aspergillus spp. obtainedusing methods described in Example 25.

FIG. 2 is a table of MIC values versus Candida spp. at 24 and 48 hoursobtained using methods described in Example 26.

FIGS. 3A and 3B are reverse phase HPLC chromatograms of a mixture ofanidulafungin and compound 1 isomers (FIG. 3A) and a purified sample ofcompound 1 (FIG. 3B). The chromatograms were obtained using the methoddescribed in Example 30.

DETAILED DESCRIPTION

The invention features echinocandin class compounds that have beenmodified such that they can exhibit (i) activity against one or morefungal species or genera; (ii) increased aqueous solubility and/oramphiphilicity; (iii) have an increased therapeutic index; (iv)suitability for topical administration; (v) suitability for intravenousadministration; (vi) have an increased volume of distribution; and/or(vii) have an increased elimination half-life.

Synthesis

The compounds of the invention include compounds of formulas (I), (II),and (III). These compounds can be synthesized, for example, as describedin the examples by coupling functionalized or unfunctionalizedechinocandin class compounds with the appropriate acyl, alkyl, hydroxyl,and/or amino groups under standard reaction conditions.

Typically, the semi-synthetic echinocandin class compounds of theinvention can be made by modifying the naturally occurring echinocandinscaffold. For example, pneumocandin B₀ is prepared by fermentationreactions; where fermentation and mixed broths produce a mixture ofproducts which are then separated to produce pneumocandin B₀, which isused in the synthesis of caspofungin (see U.S. Pat. No. 6,610,822, whichdescribes extraction of the echinocandin class compounds, such as,pneumocandin B₀, WF 11899 and echinocandin B by performing severalextraction processes; and see U.S. Pat. No. 6,610,822, which describesmethods for purifying the crude extracts).

For semi-synthetic approaches to compounds of the invention, thestereochemistry of the compound will be dictated by the startingmaterial. Thus, the stereochemistry of the unnatural echinocandinderivatives will typically have the same stereochemistry as thenaturally occurring echinocandin scaffold (representativestereochemistry is depicted in the examples) from which they arederived. Accordingly, any of the compounds shown below anidulafungin,caspofungin, or micafungin can be used as a starting material in thesynthesis of the compounds of the invention which share the samestereochemical configuration at each of the amino acid residues found inthe naturally occurring compound.

Accordingly, the echinocandin class compounds of the invention can bederived from the cyclic peptide antifungals which are produced byculturing various microorganisms.

The compounds of the invention can be synthesized, for example, usingthe methods described in the examples.

The compounds of the invention can also be used as starting materials inthe synthesis of the compounds of formula (Ib) and (IIb). For example,amine-terminating compounds can be used to prepare guanidinederivatives. The conversion of amino groups to guanidine groups can beaccomplished using standard synthetic protocols. For example, Mosher hasdescribed a general method for preparing mono-substituted guanidines byreaction of aminoiminomethanesulfonic acid with amines (Kim, K.; Lin,Y.-T.; Mosher, H. S. Tetrahedron Lett. 29: 3183, 1988). A moreconvenient method for guanylation of primary and secondary amines wasdeveloped by Bernatowicz employing 1H-pyrazole-1-carboxamidinehydrochloride;1-H-pyrazole-1-(N,N′-bis(tert-butoxycarbonyl)carboxamidine; or1-H-pyrazole-1-(N,N′-bis(benzyloxycarbonyl)carboxamidine. These reagentsreact with amines to give mono-substituted guanidines (see Bernatowiczet al., J. Org. Chem. 57: 2497, 1992; and Bernatowicz et al.,Tetrahedron Lett. 34: 3389, 1993). In addition, Thioureas andS-alkyl-isothioureas have been shown to be useful intermediates in thesyntheses of substituted guanidines (Poss et al., Tetrahedron Lett. 33:5933 1992). The compounds of formula (Ib) and (IIb) that include aheterocyclic ring can be synthesized, for example, by coupling ahydroxyalkyl or aminoalkyl substituted heterocycle with a parentechinocandin compound using those coupling methods described in theexamples.

Therapy and Formulation

The invention features compositions and methods for treating orpreventing a disease or condition associated with a fungal infection (e.g., a yeast infection) by administering a compound of the invention.

Compounds of the present invention may be administered by anyappropriate route for treatment or prevention of a disease or conditionassociated with a fungal infection. These may be administered to humans,domestic pets, livestock, or other animals with a pharmaceuticallyacceptable diluent, carrier, or excipient. When administered orally,these may be in unit dosage form, or in as a liquid oral dosage form.Administration may be topical, parenteral, intravenous, intra-arterial,subcutaneous, intramuscular, intracranial, intraorbital, ophthalmic,intraventricular, intracapsular, intraspinal, intracisternal,intraperitoneal, intranasal, aerosol, by suppositories, or oraladministration.

Therapeutic formulations may be in the form of liquid solutions orsuspensions; for oral administration formulations in the form of tabletsor capsules, syrups, or oral liquid dosage forms; intranasalformulations, in the form of powders, nasal drops; formulated as eardrops; as formulated as aerosols, or formulated for topicaladministration, such as a cream or ointment.

Methods well known in the art for making formulations are found, forexample, in “Remington: The Science and Practice of Pharmacy” (20th ed.,ed. A. R. Gennaro, 2000, Lippincott Williams & Wilkins). Formulationsfor parenteral administration may, for example, contain excipients,sterile water, or saline, polyalkylene glycols such as polyethyleneglycol, oils of vegetable origin, or hydrogenated napthalenes.Formulations for inhalation may contain excipients, for example,lactose, or may be aqueous solutions containing, for example,polyoxyethylene-9-lauryl ether, glycholate and deoxycholate, or may beoily solutions for administration in the form of nasal drops, or as agel. The concentration of the compound in the formulation will varydepending upon a number of factors, including the dosage of the drug tobe administered, and the route of administration.

The compound or combination may be optionally administered as apharmaceutically acceptable salt, such as acid addition salts; metalsalts formed by the replacement of an acidic proton with a metal, suchas an alkali or alkaline earth salts (e.g., sodium, lithium, potassium,magnesium, or calcium salts); or metal complexes that are commonly usedin the pharmaceutical industry. Examples of acid addition salts includeorganic acids such as acetic, lactic, pamoic, maleic, citric, malic,ascorbic, succinic, benzoic, palmitic, suberic, salicylic, tartaric,methanesulfonic, toluenesulfonic, or trifluoroacetic acids; polymericacids such as tannic acid, and carboxymethyl cellulose; and inorganicacid such as hydrochloric acid, hydrobromic acid, sulfuric acid,phosphoric acid. Metal complexes include zinc, and iron, among others.

Formulations for oral use include tablets containing the activeingredient(s) in a mixture with non-toxic pharmaceutically acceptableexcipients. These excipients may be, for example, inert diluents orfillers (e.g., sucrose and sorbitol), lubricating agents, glidants, andantiadhesives (e.g., magnesium stearate, zinc stearate, stearic acid,silicas, hydrogenated vegetable oils, or talc). Formulations for oraluse may also be provided in unit dosage form as chewable tablets,tablets, caplets, or capsules (i.e., as hard gelatin capsules whereinthe active ingredient is mixed with an inert solid diluent, or as softgelatin capsules wherein the active ingredient is mixed with water or anoil medium).

The compounds of the invention can be formulated with excipients thatimprove the oral bioavailability of the compound. For example, thecompounds of the invention can be formulated for oral administrationwith medium chain (C8 to C12) fatty acids (or a pharmaceuticallyacceptable salt thereof), such as capric acid, caprylic acid, lauricacid, or a pharmaceutically acceptable salt thereof, or a mixturethereof. The formulation can optionally include a medium chain (C8 toC12) alkyl alcohol, among other excipients. Alternatively, the compoundsof the invention can be formulated for oral administration with one ormore medium chain alkyl saccharides (e.g,. alkyl (C8 to C14)beta-D-maltosides, alkyl (C8 to C14) beta-D-Gulcosides, octylbeta-D-maltoside, octyl beta-D-maltopyranoside, decyl beta-D-maltoside,tetradecyl beta-D-maltoside, octyl beta-D-glucoside, octylbeta-D-glucopyranoside, decyl beta-D-glucoside, dodecylbeta-D-glucoside, tetradecyl beta-D-glucoside) and/or medium chain sugaresters (e.g., sucrose monocaprate, sucrose monocaprylate, sucrosemonolaurate and sucrose monotetradecanoate).

The formulations can be administered to human subjects intherapeutically effective amounts. Typical dose ranges are from about0.01 μg/kg to about 800 mg/kg, or about 0.1 mg/kg to about 50 mg/kg, ofbody weight per day. The preferred dosage of drug to be administered islikely to depend on such variables as the type and extent of thedisorder, the overall health status of the particular subject, thespecific compound being administered, the excipients used to formulatethe compound, and its route of administration.

The compounds of the invention can be used to treat, for example, tineacapitis, tinea corporis, tinea pedis, onychomycosis, perionychomycosis,pityriasis versicolor, oral thrush, vaginal candidosis, respiratorytract candidosis, biliary candidosis, eosophageal candidosis, urinarytract candidosis, systemic candidosis, mucocutaneous candidosis,aspergillosis, mucormycosis, paracoccidioidomycosis, North Americanblastomycosis, histoplasmosis, coccidioidomycosis, sporotrichosis,fungal sinusitis, and chronic sinusitis.

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how themethods and compounds claimed herein are performed, made, and evaluated,and are intended to be purely exemplary of the invention and are notintended to limit the scope of what the inventors regard as theirinvention.

Analytical HPLC was performed using the following column(s) andconditions: Phenomenex Luna C18(2), 5 μm, 100 Å, 2.0×150 mm, 1-99% CH₃CN(0.1% TFA) in H₂O (0.1% TFA)/15 min. Preparative HPLC was performedusing the following column: Waters Nova-Pak HR C18, 6 μm, 60 Å, 19×300mm, CH₃CN/H₂O various linear gradients and modifiers as necessary at 10mL/min.

The following abbreviations are used in the examples below: min(minutes), hr (hours), mmol (millimole), mL (milliliter), μm (micron), Å(angstrom), THF (tetrahydrofuran), DMF (dimethylformamide), TLC (thinlayer chromatography), TFA (trifluoroacetic acid), HPLC (highperformance liquid chromatography), RP (reversed phase), DIEA(diisopropylethylamine), LC/MS (liquid chromatography/massspectrometry), T_(R) (retention time on HPLC), C (Celsius), and FMOC(fluorenylmethyloxycarbonyl).

Example 1 Synthesis of Compound 1

Anidulafungin (5 mg; 0.004 mmol) dissolved in anhydrous DMSO (0.2 mL)was treated choline chloride (13 mg; 0.093 mmol) and HCl (4M in1,4-dioxane; 1.0 μL; 0.004 mmol). The resulting solution was stirred atroom temperature for 2 days and heated at 40° C. for ˜8 hr then dilutedwith water and acetonitrile and purified by preparative RP HPLC elutingwith water (0.1% TFA)/CH₃CN (0.1% TFA). The product was isolated byfreeze-drying to give 2.0 mg of compound 1 as a white solid. HPLC T_(R)10.84 min (90%). LC/MS, ESI+m/z 1225.60 [M]⁺.

Two alternative synthetic protocols are provided below.

Anidulafungin (3.00 g; 2.63 mmol) was suspended in dry THF (5 ml) andtreated with phenylboronic acid (386 mg; 3.17 mol). The mixture wasstirred until all solid dissolved (˜30 min) then for an additional 30min. THF was removed in vacuo at room temperature. The residue was againdissolved in THF and concentrated to dryness and then suspended in dryCH₃CN and concentrated to dryness to remove water. The resulting solidand N,N-dimethylethanolamine hydrochloride (9.25 g; 73.6 mmol) weremixed in dry DMSO (10 mL) until dissolved. The resulting clear viscoussolution was treated with 4M HCl in dioxane (0.33 mL). The solution wasstirred at room temperature for 3 days. The reaction was diluted withwater (10 mL), and the resulting solution was then added slowly to asolution of sodium bicarbonate (12.4 g; 2 eq. vs. amine hydrochloride)in 300 ml water with vigorous stirring. The resulting flocculentprecipitate was isolated by centrifuge. The solid was then trituratedwith 200 mL of water to give a translucent homogeneous suspension thatwas separated by centrifuge. The solids were dissolved in DMSO (15.0 mL)and treated with DIEA (0.460 ml; 2.64 mmol) was added followed by CH₃I(0.250 mL; 4.02 mmol), and the solution was stirred for 20 min at roomtemperature. Water (7 mL) was added followed by methanol (7 mL) andacetic acid (0.300 ml). The resulting solution was further diluted withwater (7 mL) and purified by preparative RP HPLC eluting withCH3CN/aqueous 0.05M ammonium acetate pH 5.0. Fractions of interest werecombined and concentrated in vacuo at 25° C. then freeze-dried to give2.33 g of compound 1 as a white solid. HPLC T_(R) 10.84 min (>98%).LC/MS, ESI+/−m/z 1225.60 [M]⁺.

Anidulafungin (0.052 g; 0.046 mmol) was suspended in dry THF (˜2 mL) andtreated with phenylboronic acid (7 mg; 0.057 mol). The mixture wasstirred until all solid dissolved (˜30 min) and then for an additional30 min. THF was removed in vacuo at room temperature. The residue wasagain dissolved in THF and concentrated to dryness and then suspended indry CH₃CN and concentrated to dryness to remove water. The resultingsolid at 0° C. was suspended in 20% TFA/CH₃CN (2.5 mL) and treatedcholine chloride (0.406 g; 2.9 mmol) and allowed to warm to roomtemperature. The solution was stirred at room temperature for 3 hr andthen overnight at 5° C. The reaction was concentrated in vacuo at roomtemperature and then diluted with methanol and water and purified bypreparative RP HPLC eluting with CH₃CN/aqueous 0.05M ammonium acetate pH5.0. Fractions of interest were combined and concentrated in vacuo at25° C. then freeze-dried to give 37 mg of compound 1 as a white solid.HPLC T_(R) 10.84 min (98%). LC/MS, ESI+/−m/z 1225.60 [M]⁺.

Example 2 Synthesis of Compound 2

N,N-Dimethylethanolamine (9.9 μL; 0.100 mmol) in anhydrous DMF wastreated with HCl (4M in 1,4-dioxane; 26.0 μL; 0.104 mmol). Anidulafungin(5 mg; 0.004 mmol) was added, and the resulting solution was stirred atroom temperature for 1 day and heated at 40° C. for 3 days. The reactionwas then diluted with water and acetonitrile and purified by preparativeRP HPLC eluting with water (0.1% TFA)/CH₃CN (0.1% TFA). The product wasisolated by freeze-drying to give 2.1 mg of compound 2 as a white solid.HPLC T_(R) 10.94 min (86%). LC/MS, ESI+m/z 1211.58 [M+H]⁺.

Example 3 Synthesis of Compound 3

Anidulafungin (5 mg; 0.004 mmol) was mixed with N-methyl-2-aminoethanolhydrochloride (0.1 g; 0.9 mmol). Anhydrous DMSO (0.1 mL) was added, andthe resulting solution was treated with HCl (4M in 1,4-dioxane; 1.0 μL;0.004 mmol) and stirred at room temperature for 4 days. The reaction wasthen diluted with water and purified by preparative RP HPLC eluting withwater (0.1% TFA)/CH₃CN (0.1% TFA). The product was isolated byfreeze-drying to give 3.1 mg of compound 3 as a white solid. HPLC T_(R)10.98 min (98%). LC/MS, ESI+m/z 1197.57 [M+H]⁺.

Example 4 Synthesis of Compound 4

Ethanolamine (6.0 μL; 0.10 mmol) in anhydrous DMF was treated with HCl(4M in 1,4-dioxane; 26.0 μL; 0.104 mmol). Anidulafungin (6.4 mg; 0.0056mmol) was added to give a clear solution which was stirred at roomtemperature for 16 days. The reaction was then diluted with water andacetonitrile and purified by preparative RP HPLC eluting with water(0.1% TFA)/CH₃CN (0.1% TFA). The product was isolated by freeze-dryingto give 2.8 mg of compound 4 as a white solid. HPLC T_(R) 10.90 min(95%). LC/MS, ESI+m/z 1183.55 [M+H]⁺.

Example 5 Synthesis of Compound 5

Anidulafungin (20 mg; 0.018 mmol) and 2-(2-aminoethoxy)ethanolhydrochloride (51 mg; 0.36 mmol) were dissolved in anhydrous DMSO (0.7mL) and treated with HCl (4M in 1,4-dioxane; 4.0 μL; 0.016 mmol). Theresulting solution was stirred at room temperature for 4 days thendiluted with water and purified by preparative RP HPLC eluting withwater (0.1% TFA)/CH₃CN (0.1% TFA). The product was isolated byfreeze-drying to give 13 mg of compound 5 as a white solid. HPLC T_(R)10.82 min (>99%). LC/MS, ESI+/−m/z 1227.6 [M+H]⁺, 1225.6 [M−H]⁻.

Example 6 Synthesis of Compound 6

2-(2-aminoethyoxy)-ethyl hemiaminal ether of anidulafungintrifluoroacetate (18 mg; 0.013 mmol) was dissolved in dry THF andconcentrated to dryness at <30° C. The solid residue was taken up inDMSO and treated with DIEA (9 μL; 0.052 mmol) followed by CH₃I (2.5 μL;0.040 mmol). The resulting solution was stirred overnight at roomtemperature then treated with additional CH₃I (1 μL; 0.016 mmol) andstirred for 2 hr longer. The solution was then diluted with water andmethanol and purified by preparative RP HPLC eluting with water (0.1%TFA)/CH₃CN (0.1% TFA). The product was isolated by freeze-drying to give9 mg of COMPOUND 6 as a white solid. HPLC T_(R) 10.92 min (>99%). LC/MS,ESI+/−m/z 1269.6 [M]⁺, 1267.6 [M−2H]⁻.

Example 7 Synthesis of Compound 7

2-(2-aminoethyl)-aminoethanol (139 mg; 1.33 mmol) in 0.5 mL of DMSO wastreated with HCl (4M in dioxane; 0.670 mL; 2.68 mmol) to give abi-phasic mixture. Anidulafungin (35 mg; 0.031 mmol) was added followedby an additional 0.5 mL of DMSO. The mixture was heated to 40° C. togive a clear solution that was heated at 35° C. overnight. The solutionwas then diluted with water and methanol and purified by preparative RPHPLC eluting with water (0.1% TFA)/CH₃CN (0.1% TFA). The product wasisolated by freeze-drying to give 11 mg of COMPOUND 7 as a white solid.HPLC T_(R) 10.28 min (>95%). LC/MS, ESI+/−m/z 1226.6 [M]+, 1224.6[M−2H]⁻.

Example 8 Synthesis of Compound 8

Anidulafungin (450 mg; 0.395 mmol) was twice suspended in acetonitrile(20 mL) and concentrated to dryness. The sample was then taken up in 20mL of anhydrous THF and concentrated to ˜10 mL. The solution under argonatmosphere was treated with phenylboronic acid (58 mg; 0.48 mmol)followed by activated 3 Å molecular sieves. The mixture was stirredslowly overnight then the supernatant was transferred to a dry flaskwith 2×5 mL THF rinses. The THF solution was concentrated to dryness thesolid residue suspended in 40 mL of dry CH₃CN. The suspension wasconcentrated to 20 mL and under argon atmosphere cooled to −10° C. andtreated with 4-methoxythiophenol (75 μL; 0.47 mmol) followed by TFA (2.4mL). The resulting mixture was stirred at −15° C. overnight then, at−10° C., quenched by slow addition of water until flocculent precipitatedeveloped. The mixture was stirred at 0° C. for 30 min then separated.Addition of water to the supernatant provided additional precipitate.The precipitates were dried in vacuo then combined and triturated withEt₂O. The solids were separated and triturated a second time with Et₂O.The isolated solids were then dried in vacuo overnight to give 393 mg ofanidulafungin hemiaminal-(4-methoxy)phenylthioether as a white powder.HPLC T_(R) 13.3 min (88%). LC/MS, ESI+/−m/z 1187.60 [M+H]⁺, 1185.58[M−H]⁻.

Anidulafungin hemiaminal-(4-methoxy)phenylthioether (21 mg; 0.017 mmol)was dissolved in 0.1 mL of 1-methyl-2-aminoethanol to give a clearsolution which was capped under argon and heated at 60° C. for 3 hr thenstirred at room temperature overnight. The reaction was diluted withmethanol, acidified by addition of TFA, further diluted with water, andpurified by preparative RP HPLC eluting with CH₃CN/H₂O and 0.1% TFA.Purified product was isolated by freeze-drying to give 22 mg of compound8 as a white solid. HPLC T_(R) 11.15 min (84%). LC/MS, ESI+/−m/z 1197.57[M+H]⁺, 1219.55 [M+Na]+, 1195.56 [M−H]⁻.

Example 9 Synthesis of Compound 9

Anidulafungin hemiaminal-(4-methoxy)phenylthioether (20 mg; 0.016 mmol)was dissolved in 0.1 mL of 1-methyl-2-(2-aminoethyoxy)ethanol to give aclear solution which was capped under argon and heated at 60° C. for 8hr then stirred at room temperature overnight. The reaction was dilutedby add with methanol and water, acidified by addition of TFA, furtherdiluted with water, and purified by preparative RP HPLC eluting withCH₃CN/H₂O and 0.1% TFA. Purified product was isolated by freeze-dryingto give 15 mg of compound 9 as a white solid. HPLC T_(R) 11.18 min(86%). LC/MS, ESI+/−m/z 1241.60 [M+H], 1239.59 [M−H]⁻.

Example 10 Synthesis of Compound 10

Anidulafungin hemiaminal-(4-methoxy)phenylthioether (21 mg; 0.017 mmol)was dissolved in 0.1 mL of mPEG₄-NH₂ to give a clear solution which wascapped under argon and heated at 50° C. overnight then at 65° C. for 2hr. The reaction was diluted with methanol and water, acidified byaddition of TFA, further diluted with water, and purified by preparativeRP HPLC eluting with CH₃CN/H₂O and 0.1% TFA. Purified product wasisolated by freeze-drying to give 13 mg of compound 10 as a white solid.HPLC T_(R) 11.26 min (88%). LC/MS, ESI+/−m/z 1329.65 [M+H]⁺, 1351.63[M+Na], 1327.64 [M−H]⁻.

Example 11 Synthesis of Compounds 11

Anidulafungin hemiaminal-(4-methoxy)phenylthioether (20 mg; 0.016 mmol)was dissolved in 0.3 mL of 2-(2-aminoethyoxy)ethanol to give a clearsolution which was capped under argon and stirred at room temperatureovernight then heated at 60° C. for 1 hr. The reaction was diluted withmethanol and water, acidified by addition of TFA, further diluted withwater, and purified by preparative RP HPLC eluting with CH₃CN/H₂O and0.1% TFA. Purified product was isolated by freeze-drying to give 15 mgof compound 11 as a white solid. HPLC T_(R) 10.83 min (94%). LC/MS,ESI+/−m/z 1227.58 [M+H]⁺, 1225.57 [M−H]⁻.

Example 12 Synthesis of Compound 12

Anidulafungin hemiaminal-(4-methoxy)phenylthioether (19 mg; 0.015 mmol)was dissolved in 0.1 mL of 2-(2-aminoethyoxy)-ethylamine to give a clearsolution which was capped under argon and heated at 60° C. for 1.5 hr.The reaction was diluted with methanol and water, acidified by additionof TFA, further diluted with water, and purified by preparative RP HPLCeluting with CH₃CN/H₂O and 0.1% TFA. Purified product was isolated byfreeze-drying to give 11 mg of compound 12 as a white solid. HPLC T_(R)10.06 min (94%). LC/MS, ESI+/−m/z 1226.60 [M+H]⁺, 1225.59 [M−H]⁻.

Example 13 Synthesis of Compound 13

Anidulafungin hemiaminal-(4-methoxy)phenylthioether (20 mg; 0.016 mmol)was dissolved in 0.1 mL of diamine to give a clear solution, which wascapped under argon and heated at 60° C. for 1.5 hr and then allowed tostir at room temperature overnight. The reaction was diluted withmethanol and water, acidified by addition of TFA, further diluted withwater, and purified by preparative RP HPLC eluting with CH₃CN/H₂O and0.1% TFA. Purified product was isolated by freeze-drying to give 11 mgof compound 13 as a white solid. HPLC T_(R) 10.06 min (92%). LC/MS,ESI+/−m/z 1270.62 [M+H]⁺, 1268.61 [M−H]⁻.

Example 14 Synthesis of Compound 14

Anidulafungin hemiaminal-(4-methoxy)phenylthioether (21 mg; 0.017 mmol)was dissolved in 0.1 mL of mono-BOC—NH—PEG₄-NH₂ to give a clear solutionwhich was capped under argon and heated at 50° C. until product:starting material ratio was ˜1:1. The reaction was treated with TFA (4mL), stirred at room temperature for ˜30 min, concentrated in vacuo,then diluted with water, and purified by preparative RP HPLC elutingwith CH₃CN/H₂O and 0.1% TFA. Purified product was isolated byfreeze-drying to give 5 mg of compound 14 as a white solid. HPLC T_(R)10.13 min (76%). LC/MS, ESI+/−m/z 1314.65 [M+H]⁺, 1312.64 [M−H]⁻.

Example 15 Synthesis of Compound 15

Anidulafungin hemiaminal-(4-methoxy)phenylthioether (49 mg; 0.039 mmol)suspended in 0.5 mL of CH₃CN at 0° C. was treated with 0.3 mL ofethylene diamine to give a clear solution which was allowed to come toroom temperature and stirred for 4 hr. The solution was diluted with 0.5mL of methanol and 2 mL of water and acidified with TFA. The solutionwas further diluted with 2 mL each of methanol and water then purifiedby preparative RP HPLC eluting with CH₃CN/H₂O and 0.1% TFA. Purifiedproduct was isolated by freeze-drying to give 30 mg of compound 15 as awhite solid. HPLC T_(R) 10.13 min (88%). LC/MS, ESI+/−m/z 1182.57[M+H]⁺, 1180.56 [M−H]⁻.

Example 16 Synthesis of Compound 16

N,N-dimethylaminoethyl anidulafungin aminal: Anidulafunginhemiaminal-(4-methoxy)phenylthioether (150 mg; 0.119 mmol) was dissolvedin 0.5 mL of N,N-dimethylethylene diamine to give a light yellowsolution which was heated at 45° C. for 18 hours. The reaction wasdiluted with 80 mL of Et₂O to precipitate product. The collected solidswere triturated with Et₂O then separated and dried in vacuo overnight togive 153 mg of N,N-dimethylaminoethyl anidulafungin aminal as anoff-white solid. HPLC T_(R) 10.37 min (76%). LC/MS, ESI+/−m/z 1210.60[M+H]⁺, 1208.60 [M−H]⁻.

N,N-dimethylaminoethyl anidulafungin aminal (153 mg; ≦0.119 mmol) inanhydrous DMSO was treated with CH₃I (8 μL; 0.129 mmol) and stirred atroom temperature overnight. The reaction was treated with additionalCH₃I (3 μL; 0.048 mmol) and DIEA (7 μL; 0.040 mmol) and stirred at roomtemp for an additional 2 hr longer. The reaction was acidified with ˜5μL of TFA, diluted with water and methanol, and purified by preparativeRP HPLC eluting with CH₃CN/H₂O and 0.1% TFA. Purified product wasisolated by freeze-drying to give 64 mg of compound 16 as a white solid.HPLC T_(R) 10.36 min (92%). LC/MS, ESI+/−m/z 1224.61 [M]⁺, 1222.61[M−2H]⁻, 1268.61 [M+formate]⁻.

Example 17 Synthesis of Compound 17

Anidulafungin hemiaminal-(4-methoxy)phenylthioether (82 mg; 0.065 mmol)was dissolved in 0.3 mL of diethylenetriamine to give a clear solutionwhich was capped under argon and heated at 60° C. for 45 min. The cooledsolution was diluted with diethyl ether to precipitate products and thesupernatant was removed. The solids were concentrated in vacuo and thentaken up in 1 mL of methanol and 1 mL of water and acidified with TFA.The resulting solution was purified by preparative RP HPLC eluting withCH₃CN/H₂O and 0.1% TFA. Purified product was isolated by freeze-dryingto give 44 mg of compound 17 as a white solid. HPLC T_(R) 9.77 min(84%). LC/MS, ESI+/−m/z 1225.61 [M+H]⁺, 1223.61 [M−H]⁻.

Example 18 Synthesis of Compound 18

Anidulafungin hemiaminal-(4-methoxy)phenylthioether (19 mg; 0.015 mmol)was dissolved in 0.3 mL of N-(2-aminoethyl)-2-aminoethanol to give aclear solution which was capped under argon and stirred at roomtemperature overnight then heated at 60° C. for 1.5 hr. The reaction wasdiluted with methanol and water, acidified by addition of TFA, furtherdiluted with water, and purified by preparative RP HPLC eluting withCH₃CN/H₂O and 0.1% TFA. Purified product was isolated by freeze-dryingto give 10 mg of compound 18 as a white solid. HPLC T_(R) 10.09 min(90%). LC/MS, ESI+/−m/z 1226.60 [M+H]⁺, 1225.59 [M−H]⁻.

Example 19 Synthesis of Compound 19

Pneumocandin B₀ hemiaminal-(4-methoxy)phenylthioether: Pneumocandin B₀(509 mg; 0.48 mmol) was twice suspended in CH₃CN (25 mL) andconcentrated in vacuo at room temperature to remove water. The samplewas then suspended in anhydrous THF (25 mL) and concentrated to dryness.The residue was taken up in anhydrous THF (25 mL) and concentrated atroom temperature to 10 mL. The resulting suspension was treated withphenylboronic acid (69 mg; 0.57 mmol) and stirred at room temperatureuntil all solid dissolved to give a slightly turbid solution. Thesolution was concentrated to dryness, and the remaining solid wassuspended in CH₃CN (25 mL). The resulting suspension was concentrated to˜10 mL, cooled to −10° C. under dry argon, and treated with4-methoxythiophenol (72 μL) followed by trifluoroacetic acid (1.4 mL)added dropwise. The mixture was stirred under argon at −15 to −20° C.for 2 days, then brought to 0° C. and treated with 25 mL of water addeddropwise to give a white suspension which was stirred at 0° C. for 20minutes then separated. The solids were triturated and re-suspended in20 mL of 25% CH₃CN/H₂O then separated and dried in vacuo. The resultingsolid was triturated with diethyl ether. The supernatant was removed,and the solids were dried in vacuo to give 430 mg of the title compoundas a dense white powder. 89% purity by HPLC, UV det. at 216-224 nm;LC/MS ESI+/−m/z 1187.59 [M+H]⁺ (calculated 1187.59), 1185.58 [M−H]⁻(calculated 1185.58), 1045.55 [M-MeOPhSH]⁻ (calculated 1045.55).

Pneumocandin B₀ hemiaminal-(4-methoxy)phenylthioether Amine:Pneumocandin B₀ hemiaminal-(4-methoxy)phenylthioether (533 mg; 0.45mmol) suspended in anhydrous THF (15 mL) was treated with phenyl boronicacid (67 mg; 0.55 mmol) followed by 3 Å molecular sieves (˜15 beads).The mixture under argon was stirred at room temperature overnight togive a clear solution which was concentrated to ˜7 mL under dry argonstream in a vented flask. The solution was then transferred to a dryflask followed by 2 THF rinses of the molecular sieves to give areaction volume of ˜10 mL. The solution under argon was cooled to 0° C.,treated with BH₃-DMS (2.0 M in THF; 1.4 mL; 2.8 mmol), and stirred at 0°C. After 2 hr, anhydrous THF (2 mL) was added to dissolve gel that hadformed. This was followed by additional BH₃-DMS (0.5 mL; 1.0 mmol). Themixture was maintained at 0° C. for 1 hr longer, then stirred at roomtemperature for ˜3 hr, then cooled to 0° C. and quenched by slowaddition of 1M HCl (1.3 mL). The quenched reaction was stored at −20° C.overnight then concentrated to ˜3 mL. The resulting mixture was dilutedwith methanol and water (˜1:1) to 20 mL then purified by preparative RPHPLC eluting with 10-70% CH₃CN/H₂O (0.1% TFA). Fractions of interestwere combined and freeze-dried to give 245 mg (42% TY) of the titlecompound as a bright white solid. 94% purity by HPLC, UV det. at 216-224nm; LC/MS ESI+/−m/z 1173.61 [M+H]⁺ (calculated 1173.61), 1031.57[M-MeOPhSH]⁻ (calculated 1031.57).

Pneumocandin hemiaminal-(4-methoxy)phenylthioether amine (20 mg; 0.016mmol) was dissolved in 2, 2′-(ethylenedioxy)bis(ethylamine) (0.1 mL).The solution was heated at 40° C. overnight and then at 60° C. for 1.5hr and then diluted with methanol (0.5 mL) and water (2 mL) andacidified with TFA. The acidified mixture was further diluted with waterand methanol then purified by preparative RP HPLC eluting with water(0.1% TFA)/CH₃CN (0.1% TFA). Product was isolated by freeze-drying togive 15 mg of compound 19 as a white solid. HPLC T_(R) 9.22 min (99%).LC/MS, ESI+/−m/z 1181.70 [M+H]+, 1179.70 [M−H]⁻.

Example 20 Synthesis of Compound 20

Pneumocandin hemiaminal-(4-methoxy)phenylthioether amine (17 mg; 0.013mmol) was dissolved in 2, 2′-oxobis(ethylamine) (0.1 mL). The solutionwas heated at 40° C. overnight and then diluted with methanol (0.5 mL)and water (2 mL) and acidified with TFA. The acidified mixture wasfurther diluted with water and methanol and then purified by preparativeRP HPLC eluting with water (0.1% TFA)/CH₃CN (0.1% TFA). Product wasisolated by freeze-drying to give 12 mg of compound 20 as a white solid.HPLC T_(R) 9.22 min (96.0%). LC/MS, ESI+/−m/z 1137.68 [M+H]⁺, 1135.67[M−H]⁻.

Example 21 Synthesis of Compound 21

Pneumocandin hemiaminal-(4-methoxy)phenylthioether amine (19 mg; 0.015mmol) was dissolved in 2-(aminoethylamino)ethanol (0.1 mL). The solutionwas heated at 60° C. for 2 hr and then diluted with methanol (0.5 mL)and water (2 mL) and acidified with TFA. The acidified mixture wasfurther diluted with water and methanol and then purified by preparativeRP HPLC eluting with water (0.1% TFA)/CH₃CN (0.1% TFA). Product wasisolated by freeze-drying to give 11 mg of compound 21 as a white solid.HPLC T_(R) 9.34 min (97.7%). LC/MS, ESI+/−m/z 1137.68 [M+H]⁺, 1135.67[M−H]⁻.

Example 22 Synthesis of Compound 22

Pneumocandin hemiaminal-(4-methoxy)phenylthioether amine (19 mg; 0.015mmol) was dissolved in 2-(aminoethylamino)ethanol (0.1 mL). The solutionwas stirred at room temperature for 1 hr, heated at 60° C. for 15 min,diluted with methanol (0.5 mL) and water (2 mL), and acidified with TFA.The acidified mixture was further diluted with water and methanol thenpurified by preparative RP HPLC eluting with water (0.1% TFA)/CH₃CN(0.1% TFA). Product was isolated by freeze-drying to give 13 mg ofcompound 22 as a white solid. HPLC T_(R) 9.12 min (93.1%). LC/MS,ESI+/−m/z 1136.69 [M+H]⁺, 1134.68 [M−H]⁻.

Example 23 In Vivo Activity Following IP Administration

The objective of these studies was to evaluate the efficacy of testcompounds in the mouse candidiasis infection model. Amounts of testarticles are uncorrected.

Inoculum Preparation

The strain, C. albicans R303 was transferred from frozen storage ontoSabauroud dextrose agar (SDA) plates and grown for ˜24 hr at 35° C. Theinoculum was prepared by transferring colonies from the plate tophosphate buffered saline (PBS) and the concentration adjusted toapproximately 10⁶ CFU/mL with the aid of a spectrophotometer. The stockwas diluted 1:9 to prepare the inoculum. Prior to each run theconcentration was verified using the dilution plate count method.

Female CD-1 mice were used in this study. The animals were approximatelyseven-weeks-old at the start of the study and weighed about 15-30 g.

Mice were made neutropenic with IP injections of cyclophosphamide (150mg/kg in 10 mL/kg) at 4 and 1 day before inoculation. Each animal wasinoculated with the appropriate concentration by injecting 0.1 mL ofinoculum into a tail vein. The test compounds were administered IP at 2hr after infection.

In a typical procedure, the kidneys were collected from four mice incontrol group 1 (untreated) at 2 hr after infection, and from anotherfour mice in control group 2 (untreated) at 24 hr after infection.Kidneys were removed aseptically from each mouse and were combined in asterile tube. An aliquot (2 mL) of sterile PBS was added to each tubeand the contents homogenized with a tissue homogenizer (Polytron 3100).Serial dilutions of the tissue homogenates were conducted and 0.1 mLaliquots were spread on SDA plates and the plates incubated at 35° C.overnight. The CFU/kidneys were determined from colony counts. Data wereanalyzed using one-way ANOVA with either the Tukey-Kramer MultipleComparisons Test or Dunnett Test (GraphPad InStat version 3.06, GraphPadSoftware, San Diego, Calif.).

The data reported below are the average of four mice. Each run includeda separate control and each is tabulated separately.

TABLE 1 Mean log₁₀ Difference from Treatment CFU/kidneys 24-h (IPadministration, mg/kg) (±SD) control Infected control - 2 h 3.27 ± 0.12Infected control - 24 h 5.20 ± 0.22 anidulafungin - 1 mg/kg 3.37 ± 0.72−1.83*** compound 1 - 5 mg/kg 0.40 ± 0.80 −4.80*** compound 2 - 5 mg/kg1.60 ± 0.35 −3.60*** compound 3 - 5 mg/kg 1.57 ± 0.20 −3.63*** compound4 - 5 mg/kg 0.73 ± 0.85 −4.47*** ns, difference not significant;***significant at P < 0.001.

TABLE 2 Mean log₁₀ Difference from Treatment CFU/kidneys 24-h (IPadministration, mg/kg) (±SEM) control Infected control - 2 h 2.92 ± 0.07Infected control - 24 h 4.87 ± 0.10 anidulafungin - 0.5 mg/kg 4.41 ±0.12 −0.47ns anidulafungin - 1.5 mg/kg 2.77 ± 0.20 −2.11***anidulafungin - 4.5 mg/kg 1.10 ± 0.38 −3.78*** compound 1 - 0.5 mg/kg2.80 ± 0.11 −2.08*** compound 1 - 1.5 mg/kg 1.83 ± 0.14 −3.05***compound 1 - 4.5 mg/kg 0.98 ± 0.33 −3.90*** compound 4 - 0.5 mg/kg 3.38± 0.10 −1.50** compound 4 - 1.5 mg/kg 0.65 ± 0.38 −4.22*** compound 4 -4.5 mg/kg 0.65 ± 0.38 −4.22*** compound 5 - 0.5 mg/kg 3.65 ± 0.10−1.22ns compound 5 - 1.5 mg/kg 0.73 ± 0.42 −4.15*** compound 5 - 4.5mg/kg 0.00 ± 0.00 −4.87*** compound 15 - 0.5 mg/kg 1.25 ± 0.45 −3.63***compound 15 - 1.5 mg/kg 0.96 ± 0.57 −3.91*** compound 15 - 4.5 mg/kg0.73 ± 0.42 −4.15*** compound 6 - 0.5 mg/kg 4.75 ± 0.19 −0.13ns compound6 - 1.5 mg/kg 1.60 ± 0.17 −3.27*** compound 6 - 4.5 mg/kg 0.00 ± 0.00−4.87*** ns, difference not significant; **significant at P < 0.01.***significant at P < 0.001.

TABLE 3 Mean log₁₀ Difference from Treatment CFU/kidneys 24-h (IPadministration, mg/kg) (±SEM) control Infected control - 2 h 2.89 ± 0.07Infected control - 24 h 5.20 ± 0.07 caspofungin - 0.5 mg/kg 0.33 ± 0.33−4.88*** caspofungin - 1.5 mg/kg 0.73 ± 0.42 −4.48*** caspofungin - 4.5mg/kg 0.00 ± 0.00 −5.20*** compound 19 - 0.5 mg/kg 3.01 ± 0.07 −2.19***compound 19 - 1.5 mg/kg 1.74 ± 0.18 −3.47*** compound 19 - 4.5 mg/kg1.17 ± 0.40 −4.03*** compound 20 - 0.5 mg/kg 1.96 ± 0.18 −3.25***compound 20 - 1.5 mg/kg 0.73 ± 0.42 −4.48*** compound 20 - 4.5 mg/kg1.45 ± 0.09 −3.75*** compound 21 - 0.5 mg/kg 1.57 ± 0.16 −3.63***compound 21 - 1.5 mg/kg 0.73 ± 0.42 −4.48*** compound 21 - 4.5 mg/kg1.17 ± 0.40 −4.03*** compound 22 - 0.5 mg/kg 1.65 ± 0.16 −3.56***compound 22 - 1.5 mg/kg 0.00 ± 0.00 −5.20*** compound 22 - 4.5 mg/kg0.65 ± 0.38 −4.55*** ns, difference not significant; ***significant at P< 0.001.

TABLE 4 Mean log₁₀ Difference from Treatment Dose CFU/kidneys 24-h(mg/kg) (mg/kg) (±SEM) control Infected control - 2 h 2.95 ± 0.07Infected control - 24 h — 5.10 ± 0.17 anidulafungin 0.5 3.81 ± 0.19−1.29** anidulafungin 1.5 1.08 ± 0.08 −4.03** compound 15 0.5 2.09 ±0.16 −3.01** compound 15 1.5 1.29 ± 0.18 −3.81** compound 7 0.5 4.33 ±0.17 −0.78* compound 7 1.5 1.55 ± 0.19 −3.55** compound 17 0.5 3.46 ±0.06 −1.64** compound 17 1.5 1.19 ± 0.12 −3.91** compound 8 0.5 4.78 ±0.15 −0.32ns compound 8 1.5 3.45 ± 0.37 −1.66** compound 9 0.5 5.04 ±0.13 −0.07ns compound 9 1.5 4.09 ± 0.23 −1.02** compound 10 0.5 4.89 ±0.06 −0.21ns compound 10 1.5 5.09 ± 0.11 −0.01ns compound 11 0.5 4.82 ±0.20 −0.29ns compound 11 1.5 2.98 ± 0.03 −2.13** compound 18 0.5 2.54 ±0.17 −2.56** compound 18 1.5 1.15 ± 0.09 −3.95** compound 12 0.5 3.20 ±0.11 −1.90** compound 12 1.5 1.60 ± 0.09 −3.50** compound 13 0.5 2.81 ±0.23 −2.29** compound 13 1.5 2.31 ± 0.19 −2.80** compound 14 0.5 4.74 ±0.17 −0.37ns compound 14 1.5 4.80 ± 0.34 −0.31ns ns, difference notsignificant; *significant at P < 0.05; **significant at P < 0.01.

TABLE 5 Mean log₁₀ Difference from Treatment Dose CFU/kidneys 24-h(mg/kg) (mg/kg) (±SEM) control Infected control - 2 h 2.98 ± 0.06Infected control - 24 h — 5.13 ± 0.15 anidulafungin 1 3.06 ± 0.18−2.07** compound 1 0.5 3.67 ± 0.07 −1.47** compound 1 1.5 1.56 ± 0.15−3.58** compound 1 4.5 0.33 ± 0.33 −4.81** compound 16 0.5 1.91 ± 0.34−3.22** compound 16 1.5 1.39 ± 0.16 −3.74** compound 16 4.5 1.05 ± 0.36−4.08** *significantat P < 0.05; **significantat P < 0.01.

CONCLUSIONS

This mouse model was used as a primary screening tool to test theefficacy of the compounds of the invention. The mice were renderedneutropenic to ensure that the observed results are attributable to thetest article and not the immune system of the mice inoculated with C.albicans, an organism known to accumulate in and infect the kidneys.

Kidneys were harvested from infected, but untreated control mice at 2hours and 24 hours after infection. The kidneys were then evaluated forthe fungal burden as measured in the number of colony forming units(CFU, reported in a log scale). As expected, untreated mice showed anincrease in the fungal burden from 2 hours to 24 hours after theinoculation with C. albicans.

Infected mice receiving one of the test articles had their kidneysremoved and evaluated after 24 hours, revealing varying levels of fungalburden that varied with the test article. The lower the CFU, the moreefficacious the compound at treating the fungal infection in thekidneys.

The compounds that perform the best are the ones that have the bestcombination of the following properties: (i) activity (i.e., an inactivecompound could not reduce the fungal burden), (ii) tissue penetration(i.e., a compound that does not get into the kidneys would not cureinfection there), and (iii) half-life (e.g., a compound with a shorthalf-life might not show efficacy at 24 hours).

Based upon these studies we conclude that (a) compounds 8, 9, 10, and 14performed poorly (i.e., did not have the right combination of propertiesuseful for treating C. albicans infections in this assay); (b) compounds2, 3, 11, and 13 performed moderately, demonstrating some ability tocontrol the C. albicans infection; and (c) compounds 1, 4, 5, 6, 7, 12,15, 16, 17, 18, 19, 20, 21, and 22 performed strongly, dramaticallyreducing the level of C. albicans CFUs found in the kidneys of the mice.

Example 24 Pharmacokinetics in Beagle Dogs

The test articles were administered to beagle dogs weighingapproximately 6-10 kg. Each test article was dosed at 1.4 mg/kg inaqueous saline (with or without 0.5% Tween) over course of 1-10 minutes.Diphenhydramine was kept on hand in case the dogs demonstrated ahistamine resonse. The dogs were fasted at least 12 hours prior to eachdosing and offered food after the 4-hour blood sample was taken; waterwas withheld for 1 hour prior to and 4 hours following each dosingevent. The dose for each animal was based on its most recent bodyweight. The test article was injected intravenously via a catheterplaced in the cephalic vein as a slow bolus.

Blood was collected via the jugular vein. All blood samples (˜1 mL each)were collected into K₃EDTA tubes. Following blood collection, thesamples were immediately inverted several times and were held on wet icepending centrifugation. The samples were centrifuged within ˜30 minutesof collection under refrigeration (˜5° C. for ˜10 minutes at ˜2000 g) toobtain plasma. The plasma was frozen immediately on dry ice afterseparation. The plasma samples were stored at approximately −70° C.until analysis.

Plasma (100 μL) was precipitated with 400 μL of 0.1% formic acid inacetonitrile containing the internal standard (100 ng/mL pneumocandin).The samples were then capped and vortexed for about 30 seconds followedby centrifugation at 14,000 rpm at room temperature for 10 minutes.Following centrifugation 200 μL of supernatant was transferred toplastic autosampler vials containing 200 μL of 0.1% formic acid in waterand vortexed. Samples were then analyzed by LCMSMS.

All pharmacokinetic calculations were performed using WinNonlin version4.1 (Pharsight Corp) by noncompartmental analysis. The results areprovided in Table 6, below.

TABLE 6 PK Values¹ T^(1/2) Vz, Cl Compound (hr) (mL/Kg) (mL/hr/kg)anidulafungin 11.52 ± 0.75 779 ± 30.4 47.1 ± 1.92 Compound 18 21.03 ±1.16 687 ± 58.4 22.6 ± 1.08 Compound 16  27.6 ± 1.11 874 ± 63.9 21.9 ±0.84 Compound 5 33.66 ± 3.28 627 ± 13.9 13.1 ± 0.94 Compound 1  53.1 ±3.93 1360 ± 61.9  18.06 ± 1.46  ¹All values are the mean ± SEM; n = 4beagle dogs.

The observed half-life of anidulafungin was approximately 12 hours,which is consistent with previously reported values.

Compound 1 was found to have a surprisingly large volume of distributionand a surprisingly long circulating half-life. These pharmacokineticproperties may provide advantages such as decreased dosing amount,decreased dosing frequency, and/or improved efficacy in thetreatment/prevention of some fungal infections.

The large volume of distribution observed for compound 1 is consistentwith the distribution of this compound into certain tissues, such askidney, liver, lung, and/or spleen. The large volume of distributionobserved for compound 1 can have clinical significance for the use ofthis compound in treating infections localized in these tissues.

Example 25 In Vitro Activity: MEC and MIC Values Versus Aspergillus Spp

MEC and MIC values (μg/mL) of anidulafungin, caspofungin, amphotericinB, compound 1, and compound 16 against various Aspergillus species inthe presence and absence of 50% human serum were obtained as follows.

Test organisms were obtained from the American Type Culture Collection(Manassas, Va.). The isolates were maintained at −80° C., then thawedand sub-cultured prior to testing.

The MIC assay method followed the procedure described by the Clinicaland Laboratory Standards Institute (see Clinical and LaboratoryStandards Institute (CLSI). Reference Method for Broth DilutionAntifungal Susceptibility Testing of Yeasts; Approved Standard, ThirdEdition. CLSI document M27-A3 [ISBN 1-56238-666-2]. Clinical andLaboratory Standards Institute, 940 West Valley Road, Suite 1400, Wayne,Pa. 19087-1898 USA, 2008; and Clinical and Laboratory StandardsInstitute (CLSI). Reference Method for Broth Dilution AntifungalSusceptibility Testing of Filamentous Fungi; Approved Standard, SecondEdition. CLSI document M38-A2 [ISBN 1-56238-668-9]. Clinical andLaboratory Standards Institute, 940 West Valley Road, Suite 1400, Wayne,Pa. 19087-1898 USA, 2008) and employed automated liquid handlers toconduct serial dilutions and liquid transfers. The wells in columns 1-12in standard 96-well microdilution plates were filled with 150 μL ofDMSO. These would become the ‘mother plates’ from which ‘daughter’ ortest plates would be prepared. The drugs (150 μL at 80× the desired topconcentration in the test plates) were dispensed into the appropriatewell in column 1 of the mother plates and mixed. Serial 1:1 dilutionswere made through Column 11 in the “mother plate”. The wells of column12 contained no drug and were the organism growth control wells. Thedaughter plates were loaded with 185 μL per well of RPMI or RPMIsupplemented with 50% human serum. The daughter plates were prepared bytransferring 5 μL of drug solution from each well of a mother plate toeach corresponding well of each daughter plate.

Standardized inoculum of Aspergillus was prepared per CLSI methods. 2 mLof 0.85% saline was dispensed onto an agar slant. Using a swab, asuspension was made. After a short time to allow the heavy particles tosettle out, a small quantity of the supernatant was dispensed into RPMIand the suspension adjusted to equal 0.5 McFarland turbidity. Dilutionswere made for each isolate in RPMI to reach the concentration of cellsdescribed in CLSI methodology. The inoculum was dispensed into sterilereservoirs divided by length, and used to inoculate the plates. Todaughter plates 10 μL of standardized inoculum was delivered into eachwell. Thus, the wells of the daughter plates ultimately contained 185 μLof broth, 5 μL of drug solution, and 10 μL of inoculums.

Plates were covered with a lid, placed in plastic bags, and incubated at35° C. The Aspergillus plates were incubated for 48 h before reading.The microplates were viewed from the bottom using a plate viewer. Foreach mother plate, an un-inoculated solubility control plate wasobserved for evidence of drug precipitation.

Both an MIC and Minimal Effective Concentration (MEC) value wasrecorded. The MEC value is applied specifically to echinocandins whentesting filamentous fungi. While the MIC value is the amount of drugthat inhibits visible growth of the organism, the MEC value is thelowest concentration of drug that leads to the growth of small, rounded,compact hyphal forms as compared to the hyphal growth seen in the growthcontrol well.

MEC values, which typically differ dramatically from MIC values for thisclass of antifungal agents, are the measure that should be used fordetermining susceptibility of filamentous fungi to echinocandins. Thegrowth of the Aspergillus strains in each well was compared with that ofthe growth control at 48 hr.

The MEC and MIC values are provided in FIG. 1. This data shows that,relative to anidulafungin, compounds 1 and 16 retain their activityagainst Aspergillus strains. Thus, some modifications were made thatproduced profound pharmacokinetic effects without restricting activityagainst Aspergillus spp.

Example 26 In Vitro Activity: MIC Values Versus Candida Spp. at 24 and48 Hours

MIC values (μg/mL) of anidulafungin, caspofungin, amphotericin B,compound 1, and compound 16 against various Candida species in thepresence and absence of 50% human serum were obtained as follows.

Test organisms were obtained from the American Type Culture Collection(Manassas, Va.). The isolates were maintained at −80° C., then thawedand sub-cultured prior to testing.

The MIC assay method followed the procedure described by the Clinicaland Laboratory Standards Institute and employed automated liquidhandlers to conduct serial dilutions and liquid transfers. The wells incolumns 1-12 in standard 96-well microdilution plates were filled with150 μL of DMSO. These would become the ‘mother plates’ from which‘daughter’ or test plates would be prepared. The drugs (150 μL at 80×the desired top concentration in the test plates) were dispensed intothe appropriate well in column 1 of the mother plates and mixed. Serial1:1 dilutions were made through Column 11 in the “mother plate”. Thewells of column 12 contained no drug and were the organism growthcontrol wells. The daughter plates were loaded with 185 μL per well ofRPMI or RPMI supplemented with 50% human serum. The daughter plates wereprepared by transferring 5 μL of drug solution from each well of amother plate to each corresponding well of each daughter plate.

Standardized inoculum of Candida was prepared per CLSI methods. For theCandida isolates, colonies were picked from the streak plate and asuspension was prepared in RPMI. Dilutions were made for each isolate inRPMI to reach the concentration of cells described in CLSI methodology.The inoculum was dispensed into sterile reservoirs divided by length,and used to inoculate the plates. To daughter plates 10 μL ofstandardized inoculum was delivered into each well. Thus, the wells ofthe daughter plates ultimately contained 185 μL of broth, 5 μL of drugsolution, and 10 μL of inoculums.

Plates were covered with a lid, placed in plastic bags, and incubated at35° C. The Candida isolates were read after 24 h incubation and again at48 h. The microplates were viewed from the bottom using a plate viewer.For each mother plate, an un-inoculated solubility control plate wasobserved for evidence of drug precipitation.

For Candida species, the Minimal Inhibitory Concentration (MIC) was readper CLSI guidelines. The MIC was defined as the lowest concentration ofan antifungal agent that substantially inhibits growth of the organismas detected visually (MIC values are provided in FIG. 2).

This data shows that, relative to anidulafungin, compounds 1 and 16retain their activity against Candida strains. Thus, some modificationswere made that produced profound pharmacokinetic effects withoutrestricting activity against Candida spp.

Serum is known to differentially alter the antifungal properties ofechinocandin drugs (see Paderu et al., Antimicrob Agents Chemother.51:2253 (2007)). Compounds 1 and 16 were found to have superior activityagainst a strain of Candida glabrarata in 50% human serum in comparisonto the performance of anidilafungin under these same conditions. Thisdifference in activity can be clinically relevant to the use of thesecompounds for the treatment of certain blood stream infections.

Example 27 Amphiphilicity of Compound 1

The solubility of compound 1 (acetate salt) was measured in aqueousbuffers of varying pH to assess this compound's suitability forformulation in an aqueous carrier for administration by injection (e.g.,intravenous or intramuscular injection).

The results are provided in Table 7 (below) along with anidulafungin asa comparison. Compound 1 was found to have dramatically greater aqueoussolubility than anidulafungin over a broad pH range.

TABLE 7 Solubility (mg/mL)¹ pH Anidulafungin Compound 1 1 (0.1M HCl)<0.01 >15 3 (0.01M formate buffer) <0.01 >15 4.6 (0.1M acetate buffer)<0.01 >15 5.6 (0.1M acetate buffer) <0.01 >15 7.4 (0.1M phosphatebuffer) <0.01 0.05 8.5 (0.01M TRIS buffer) <0.01 >15 0.9% saline <0.014.6 ¹All measurements made at ambient temperature

The solubility of compound 1 (acetate salt) was also measured innon-aqueous solvents to assess this compound's suitability forformulation in non-aqueous carriers. The results are provided in Table 8(below).

TABLE 8 Solubility (mg/mL) pH Anidulafungin Compound 1 Propyleneglycol(PG) >17.2 >18.4 Ethanol (EtOH) >17.6 >13.7 Glycerol 1.5 >19.4PEG400 >26.8 >34.1

Example 28 Synthesis of Compound 23

Anidulafungin (20 mg; 0.018 mmol) in CH₃CN (10 ml) was treated withphenylboronic acid (2.5 mg; 0.021 mmol) and the mixture was stirred for30 minutes. The resulting solution was concentrated in vacuo to dryness,and the solids were taken up in a solution of DMSO (0.3 mL) and1,3-diamino-2-propanol hydrochloride. The mixture was titrated with HCl(4M in dioxane) until acidic on wet pH paper. The resulting solution washeated at 40-45° C. for 8 days then diluted with water and purified bypreparative RP HPLC eluting with water (0.1% TFA)/CH₃CN (0.1% TFA). Theproduct was isolated by freeze-drying to give 33 mg of compound 23 as awhite solid. HPLC T_(R) 11.28 min (93%). LC/MS, ESI+/−m/z 1212.58 [M+H],1210.57 [M−H]⁻.

Example 29 Synthesis of Compound 24

Anidulafungin hemiaminal-(4-methoxy)phenylthioether (20 mg; 0.016 mmol)was mixed with serinol (114 mg) and dry DMSO (15 μL). The mixture wascapped under argon and heated to 70° C. for 2.5 hours. The reaction wasdiluted with methanol and water, acidified by addition of TFA, furtherdiluted with water, and purified by preparative RP HPLC eluting withCH₃CN/H₂O and 0.1% TFA. Purified product was isolated by freeze-dryingto give 13 mg of compound 24 as a white solid. HPLC T_(R) 10.71 min (94%@220 nm). LC/MS, ESI+/−m/z 1213.57 [M+H]⁺, 1211.56 [M−H]⁻.

Example 30 Separation of Isomers

Compound 1 purified by preparative RP HPLC eluting with CH₃CN/H₂O and0.1% TFA was found to be a mixture of isomers (see FIGS. 3A and 3B). Thetwo isomers observed are believed to differ in stereochemistry where thecholine substituent is attached to the anidulafungin starting material(see the isomers depicted below).

Other Embodiments

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each independent publication or patent application was specificallyand individually indicated to be incorporated by reference.

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure that come within known or customary practice withinthe art to which the invention pertains and may be applied to theessential features hereinbefore set forth, and follows in the scope ofthe claims.

Other embodiments are within the claims.

What is claimed is:
 1. A compound having the formula:

or a pharmaceutically acceptable salt thereof.
 2. The compound of claim1, wherein said compound is an acetate salt.
 3. The compound of claim 1,wherein said compound is a chloride salt.
 4. A pharmaceuticalcomposition comprising the compound of claim 1, or a pharmaceuticallyacceptable salt thereof, and a pharmaceutically acceptable excipient. 5.The pharmaceutical composition of claim 4, wherein said compound is anacetate salt.
 6. The pharmaceutical composition of claim 4, wherein saidcompound is a chloride salt.
 7. The pharmaceutical composition of claim4, wherein said pharmaceutical composition is formulated for oraladministration.
 8. The pharmaceutical composition of claim 4, whereinsaid pharmaceutical composition is formulated for intravenousadministration.
 9. The pharmaceutical composition of claim 4, whereinsaid pharmaceutical composition is formulated for topicaladministration.
 10. The pharmaceutical composition of claim 4, whereinsaid pharmaceutical composition is formulated for subcutaneousadministration.
 11. The pharmaceutical composition of claim 4, whereinsaid pharmaceutical composition is formulated for inhalation.
 12. Amethod of treating a fungal infection in a subject, said methodcomprising administering to said subject a pharmaceutical composition ofclaim 4 in an amount sufficient to treat said infection.
 13. The methodof claim 12, wherein said pharmaceutical composition is administeredorally.
 14. The method of claim 12, wherein said pharmaceuticalcomposition is administered intravenously.
 15. The method of claim 12,wherein said pharmaceutical composition is administered topically. 16.The method of claim 12, wherein said pharmaceutical composition isadministered subcutaneously.
 17. The method of claim 12, wherein saidpharmaceutical composition is administered by inhalation.
 18. The methodof claim 12, wherein said pharmaceutical composition is administered totreat a blood stream infection or tissue infection in said subject. 19.The method of claim 12, wherein said infection is selected from tineacapitis, tinea corporis, tinea pedis, onychomycosis, perionychomycosis,pityriasis versicolor, oral thrush, vaginal candidosis, respiratorytract candidosis, biliary candidosis, eosophageal candidosis, urinarytract candidosis, systemic candidosis, mucocutaneous candidosis,aspergillosis, mucormycosis, paracoccidioidomycosis, North Americanblastomycosis, histoplasmosis, coccidioidomycosis, sporotrichosis,fungal sinusitis, or chronic sinusitis.
 20. The method of claim 12,wherein said fungal infection is an infection of Candida albicans, C.parapsilosis, C. glabrata, C. guilliermondii, C. krusei, C. tropicalis,C. lusitaniae, Aspergillus fumigatus, A. flavus, A. terreus, A. niger,A. candidus, A. clavatus, or A. ochraceus.
 21. A method of preventing afungal infection in a subject, said method comprising administering tosaid subject a pharmaceutical composition of claim 3 in an amountsufficient to prevent said infection.
 22. The method of claim 21,wherein said pharmaceutical composition is administered intravenously.23. The method of claim 22, wherein said subject is being prepared foran invasive medical procedure, said subject is immunocompromised, orsaid subject is undergoing long term antibiotic therapy.
 24. A method ofpreventing, stabilizing, or inhibiting the growth of fungi, or killingfungi, said method comprising contacting said fungi or a sitesusceptible to fungal growth with a compound of claim 1.